Antitumor active substances

ABSTRACT

By extracting hot water-insoluble and ethanol-insoluble components of the fruit body of Agaricus blazei Murill belonging to the genus Agaricus with 5% ammonium oxalate aqueous solution, degrading the extract with hydrochloric acid and then purifying the acid-degraded product through gel permeation and affinity chromatography, there are obtained the antitumor-active substances having a weight-average molecular weight of 38×10 4  daltons and a dispersion degree of 2.3, a weight-average molecular weight of 29×10 4  daltons and a dispersion degree of 7.3, a weight-average molecular weight of 2.4×10 4  daltons and a dispersion degree of 4.1, and a weight-average molecular weight of 2.0×10 4  daltons and a dispersion degree of 3.6. These substances exhibit a significant antitumor effect against solid tumor, when determined by gel permeation.

FIELD OF THE INVENTION

The present invention relates to a substance having an antitumoractivity and, an antitumor composition and a health food, comprising thesame. More particularly, the present invention relates to a substancehaving an antitumor activity, which substance is obtainable bycollecting the extract residues with ethanol from the fruit body ofAgaricus blazei Murill belonging to the genus Agaricus, degrading theresidues with an acid and then purifying the acid-degraded product. Thepresent invention further relates to an antitumor composition and ahealth food comprising such an antitumor-active substance.

BACKGROUND ART

Agaricus blazei Murill belonging to the genus Agaricus and also calledhimemaitsutake mushroom is a kind of mushrooms that grows wild mainly inthe mountainous region of the southeastern Brazil, Sao Paulo. Thenatives have long been using Agaricus blazei Murill as an ediblemushroom.

In these years, Agaricus blazei Murill has become widely cultured alsoin Japan to make its extracts available for the treatment of diabetesand hypertension.

Various investigations have been made to explore substances having anantitumor activity. It is reported that polysaccharides with anantitumor activity are obtained, for example, by extracting the fruitbody or mycelium of Agaricus blazei Murill with an aqueous medium(Japanese Patent KOKAI (Laid-Open) Nos. 55-74797, 64-67194, 64-67195,55-108292, etc.). It is also reported that a nucleic acid componenthaving an antitumor activity is extracted from the fruit body ofhimematsutake mushroom (Japanese Patent KOKAI (Laid-Open) No. 64-66127).These substances having an antitumor activity are all extracted from thecomponents soluble in an aqueous medium or soluble in hot water.

On the other hand, Japanese Patent Application KOKAI No. 2-78630discloses that a proteoglycan having an antitumor activity was isolatedfrom the extract residue of the Agaricus blazei Murill fruit body withhot water. More specifically, it is reported that the Agaricus blazeiMurill fruit body was extracted with hot water to remove water-solublecomponents, the resulting residue was further extracted with hot 1%ammonium oxalate aqueous solution; then the extract residue gave theproteoglycan having an antitumor activity.

The substances mentioned above are obtained either from the componentssoluble in an aqueous medium or soluble in hot water, or from theextract residue with hot water. Thus these substances are all derivedfrom the components insoluble in hot 1% ammonium oxalate aqueoussolution.

On the other hand, the present inventors found that a substance havingan antitumor activity was obtained from the extract residue of theAgaricus blazei Murill fruit body with hot water followed by furtherextraction with hot 1% ammonium oxalate aqueous solution. JapanesePatent Application No. 4-160924 (Japanese Patent KOKAI (Laid-Open) No.6-9423) is directed to the substance.

However, this substance is not satisfactory for the treatment of solidtumor since its antitumor activity is not sufficiently potent.

Either the above substance obtained from the components of the Agaricusblazei Murill fruit body soluble in an aqueous medium or soluble in hotwater or the substance insoluble in hot 1% ammonium oxalate aqueoussolution which is obtained from the extract residue with hot water isyet insufficient in their antitumor activity.

DESCRIPTION OF THE INVENTION

The present inventors have found that a substance having a potentantitumor activity can be obtained by treating the Agaricus blazeiMurill fruit body with hot ethanol, collecting the extract residues,degrading the residues with an acid, preferably after extracting withammonium oxalate, or directly without ammonium oxalate extraction, andthen purifying the acid degradation product. The inventors have furtherfound that the substance is useful as an active ingredient of anantitumor composition and of a health food. The present invention hasthus been accomplished.

The present invention relates to a substance having an antitumoractivity, which is obtainable from the fruit body of Agaricus blazeiMurill belonging to the genus Agaricus by acid degradation of thecomponents of the fruit body which are insoluble in hot water andinsoluble in ethanol, followed by purification; the substance beingpreferably obtainable from the fruit body by extracting the insolublecomponents with an ammonium oxalate aqueous solution, degrading theextract with an acid and then purifying the acid-degraded product, whichsubstance has a weight-average molecular weight of 38×10⁴ daltons and adispersion degree of 2.3, an average molecular weight of 29×10⁴ daltonsand a dispersion degree of 7.3, an average molecular weight of 2.4×10⁴daltons and a dispersion degree of 4.1, or an average molecular weightof 2.0×10⁴ daltons and a dispersion degree of 3.6, when measured by gelpermeation.

The present invention further relates to an antitumor compositioncomprising as an active ingredient the antitumor-active substancedescribed above.

The present invention further relates to a health food comprising as anactive ingredient the antitumor-active substance described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a profile of chromatogram obtained when the aciddegradation product of the Agaricus blazei Murill fruit body wassubjected to gel permeation.

FIG. 2 shows a profile obtained when the high molecular componentresulting from the gel permeation shown in FIG. 1 was subjected toaffinity chromatography (DEAE fraction).

FIG. 3 shows a profile obtained when the low molecular componentresulting from the gel permeation shown in FIG. 2 was subjected toaffinity chromatography (DEAE fraction).

FIG. 4 shows a profile of chromatogram obtained when theantitumor-active substance of the present invention was subjected to gelpermeation.

FIG. 5 shows an IR spectrum of the antitumor-active substance of thepresent invention.

FIG. 6A and FIG. 6B show a one-dimensional proton NMR spectrum of theantitumor-active substance of the present invention.

FIG. 7 shows an antitumor activity of the antitumor-active substanceaccording to the present invention when mice were inoculated with Meth-Atumor cells intradermally in the right flank.

FIG. 8 shows an antitumor activity of the antitumor-active substanceaccording to the present invention when mice were inoculated with Meth-Atumor cells intradermally in the left flank.

BEST MODE FOR CARRYING OUT THE INVENTION

Agaricus blazei Murill belonging to the genus Agaricus is already knownwidely and has been deposited in Fermentation Research Institute of theAgency of Industrial Science & Technology of Japan under Accession No.4731. Known species other than the deposited Agaricus blazei Murill mayof course be employed in the present invention.

To obtain the antitumor-active substances of the present invention fromthe fruit body of Agaricus blazei Murill, the following procedures areused.

The fruit body may be fresh or dry. In general, the fresh fruit body iscut into thin strips and the dry one is finely grounded for use.

First, the fruit body is treated with 75-90% hot ethanol, preferably80-85%, e.g., 80% hot ethanol to remove soluble components. The residuesare collected. The temperature is generally kept at about 80° C. Thetreatment is generally carried out for 6 to 24 hours, preferably about18 to 22 hours, though the treating time may vary depending upon anamount of the fruit body to be treated. By such a treatment, lowmolecular organic compound components are removed.

Subsequently, the resulting residue is treated with hot water to removecomponents soluble in hot water. The residues are again collected,whereby neutral and acidic polysaccharides soluble in hot water areremoved. The temperature of hot water used for the treatment rangesgenerally from 80 to 100° C. The time required for the treatment isgenerally between 6 and 24 hours, preferably between 18 and 22 hours.

The collected residues are then freeze-dried and used for the next stepof acid degradation. Preferably, the collected residues are extractedwith 1-5%, preferably 5% hot ammonium oxalate aqueous solution torecover the components soluble in the ammonium oxalate aqueous solution.The extraction is normally carried out while boiling the ammoniumoxalate aqueous solution. The thus recovered components are collectedand concentrated. After the concentration, ammonium oxalate is subjectedto gel permeation for desalting and concentration, followed by freezedrying.

The freeze-dried product is then subjected to acid degradation. For theacid degradation, strong acids such as hydrochloric acid, sulfuric acid,nitric acid, etc. may preferably be used. Of these acids, hydrochloricacid is particularly preferred. More specifically, the freeze-driedproduct is dissolved in 1N hydrochloric acid and the solution is allowedto stand overnight at room temperature, whereby acid degradation can beeffected.

Following the acid degradation, the degradation product is neutralizedwith, e.g., 1N sodium hydroxide aqueous solution. The resulting aqueoussolution is centrifuged to remove unwanted matters. The supernatant isdesalted and concentrated through a ultrafiltration membrane,freeze-dried and then purified.

The freeze-dried acid degradation product thus obtained may be purifiedby gel permeation and affinity chromatography. Gel permeation may beperformed using, e.g., GPC column or TSKgel G 5000 PW connected withTSKgel G 3000PW (each 21.5 mm×300 mm, made bay Toso Co., Ltd.). The aciddegradation product is separated by gel permeation into a high molecularfraction with a molecular weight of approximately 10⁵ to 10⁶ daltons anda low molecular fraction with a molecular weight of approximately 5×10³to 5×10⁴ daltons.

Next, the high and low molecular fractions thus obtained are subjectedto affinity chromatography, respectively, for purification. Thus, theantitumor-active substance having a weight-average molecular weight of29×10⁴ daltons and a dispersion degree of 7.3, and the antitumor-activesubstances having a weight-average molecular weight of 2.4×10⁴ daltonsand a dispersion degree of 4.1 and having a weight-average molecularweight of 2.0×10⁴ daltons and a dispersion degree of 3.6 may be obtainedfrom the high and low molecular fractions, respectively.

The affinity chromatography may be performed using, e.g., a DEAE columnor TSKgel DEAE-5PW (21.5 mm×150 mm×2, made bay Toso Co., Ltd.). Theantitumor-active substance having a weight-average molecular weight of29×10⁴ daltons and a dispersion degree of 7.3 according to the presentinvention may be obtained by adsorbing the high molecular fractiondescribed above to a DEAE column and then eluting with 0.5M NaCl aqueoussolution. The antitumor-active substance having a weight-averagemolecular weight of 2.4×10⁴ daltons and a dispersion degree of 4.1 maybe obtained by adsorbing the low molecular fraction described above to aDEAE column and then eluting with 0.2M NaCl aqueous solution. Theantitumor-active substance having a weight-average molecular weight of2.0×10⁴ daltons and a dispersion degree of 3.6 may be obtained byeluting with 0.5M NaCl aqueous solution.

The antitumor-active substance having a weight-average molecular weightof 29×10⁴ daltons and a dispersion degree of 7.3 according to thepresent invention has an ivory white color and is cotton-like, highlyhygroscopic and water-soluble. The antitumor-active substance having aweight-average molecular weight of 2.4×10⁴ daltons and a dispersiondegree of 4.1 and the antitumor-active substance having a weight-averagemolecular weight of 2.0×10⁴ daltons and a dispersion degree of 3.6 arebrown, hygroscopic and water-soluble.

The antitumor-active substance having a weight-average molecular weightof 29×10⁴ daltons and a dispersion degree of 7.3 has a small absorptionat UV of 280 nm; it is thus considered that the substance would bemainly composed of polysaccharides or contain in part someproteoglycans. The antitumor-active substance having a weight-averagemolecular weight of 2.4×10⁴ daltons and a dispersion degree of 4.1 has aslight absorption at UV of 280 nm; it is thus considered that thesubstance would be mostly composed of polysaccharides. Theantitumor-active substance having a weight-average molecular weight of2.0×10⁴ daltons and a dispersion degree of 3.6 has a considerableabsorption at UV of 280 nm; it is thus considered that the substancewould be a complex mainly composed of proteins, to a part of whichpolysaccharides are conjugated.

The antitumor-active substance having a weight-average molecular weightof 38×10⁴ daltons and a dispersion degree of 2.3 according to thepresent invention may be obtained by further purifying the aforesaidantitumor-active substance having a weight-average molecular weight of29×10⁴ daltons and a dispersion degree of 7.3 by gel permeation using,e.g., a GPC column for desalting and concentration.

The antitumor-active substance having a weight-average molecular weightof 38×10⁴ daltons and a dispersion degree of 2.3 in accordance with thepresent invention is ivory white, cotton-like, highly hygroscopic andwater-soluble. Observation of one-dimensional proton spectrum at theanomeric proton region of the antitumor-active substance of the presentinvention through measurements of ¹ H-NMR and ¹³ C-NMR indicates thatthere are peaks at 5.27 ppm and 4.51 ppm. The peaks observed at 5.27 ppmand 4.51 ppm designate 1-4-α-glucan and 1-6β-glucan, respectively.Integration of the peaks observed by NMR reveals that a ratio of1-4-α-glucan to 1-6-β-glucan is 4:1. Therefore, the antitumor-activesubstance of the present invention is a polysaccharide mainly composedof 1-4-α-glucan and 1-6-β-glucan. In addition, IR analysis of theantitumor-active substance of the present invention indicates that therean absorption appears at 881 cm⁻¹. Thus, its specific rotary power isfound to be [α]²⁰ _(D) +121°.

The antitumor-active substances of the present invention exhibit apotent antitumor activity against, e.g., fibrosarcoma-derived Meth-A.Among solid cancers, Meth-A tumor cells are known to be most resistantto chemotherapeutic agents (Biotherapy, 3(2), 557 (1989); Biotherapy,4(4), 915 (1990); GAN-TO-KAGAKU RYOHO (Cancer and Chemotherapy), 18(11),1812 (1991)). It is therefore expected that the substances of thepresent invention will be sufficiently effective to the solid cancers.

Where the antitumor-active substances of the present invention are usedfor therapy, the substances are administered orally or parenterally. Fororal administration, tablets, capsules, granules, etc. are used. Thesepharmaceutical preparations may be prepared in a conventional manner.Parenteral preparations may also be prepared in a conventional manner,e.g., by dissolving or dispersing the antitumor-active substance in avehicle for injection conventionally used.

A dose of the antitumor-active substances of the present invention mayvary depending upon kinds of tumor, routes for administration, etc. butranges normally from 5 to 100 mg/kg body weight.

The antitumor-active substance of the present invention may also beincorporated in a health food as an active ingredient. Where theantitumor-active substance of the present invention is used in a healthyfood, dried raw materials are purified by the procedures describedabove, freeze-dried and incorporated into a food in a predeterminedratio, which is provided as foodstuffs. For example, theantitumor-active substances of the present invention may be used as afurikake or tastily seasoned dried food, as an ingredient of tea pack orin a capsule form. Furthermore, the freeze-dried or concentratedsubstance may be incorporated in dairy products, oil-and-fat products,seasonings, cakes, fruit juice, beverage, etc., for use. When added tothese products, the amount of the substance of the present inventionadded to a health food is generally from 0.001 to 0.1 wt %.

By subjecting the extract of the hot water-insoluble andethanol-insoluble components from the fruit body of Agaricus blazeiMurill with 1-5% ammonium oxalate aqueous solution to acid degradation,the substances having a potent antitumor activity against solid tumorcan be obtained.

Hereinafter, the present invention will be described in more detail,with reference to Examples.

EXAMPLE 1

Preparation of the antitumor-active substances

(1) The fruit body (30 kg) of dried himematsutake mushroom is roughlyground into 5 mm or less. To 30 kg of the fruit body 270 liters of 80%v/v ethanol is added. The mixture is extracted while heating to refluxfor 22 hours. After solid-liquid separation, 270 liters of 80% v/vethanol is added to the residue. The resulting mixture is treated asdescribed above. The procedure is repeated 3 times.

(2) After 270 liters of purified water is added to the extract residue(1) above, the mixture is extracted while heating to reflux for 22hours, followed by solid-liquid separation. Next, 270 liters of purifiedwater is added to the residue. The mixture is then treated as describedabove. The procedure is repeated 3 times. (3) After 270 liters of 5%ammonium oxalate aqueous solution is added to the extract residue (2)above, the mixture is extracted while heating to reflux for 22 hours.After solid-liquid separation, the resulting aqueous solution wasconcentrated and 270 liters of 5% ammonium oxalate aqueous solution isadded to the residue. The mixture is then treated as described above.The procedure is repeated 3 times. The extract of 800 liters in total isconcentrated to 80-100 liters.

(4) The concentrated solution is filtered through a filter paper andthen desalted/concentrated through a ultrafiltering membrane (fractionalmolecular weight of 10000). The concentrate is freeze-dried anddissolved in 1N HCl. After allowing to stand for 24 hours, the solutionis neutralized with 1N NaOH aqueous solution to become pH of 7. Thesolution is centrifuged to remove unwanted matters. The supernatant isdesalted and concentrated through a ultrafiltering membrane (fractionalmolecular weight of 10000) followed by freeze-drying.

(5) Following the acid degradation (4) described above, the purified drypowders are dissolved in 0.2M NaCl in a ratio of 10 mg/ml. After thepowders are dissolved, the solution is centrifuged and the supernatantis filtered through a membrane filter of 0.8 μm.

The filtered supernatant is passed through a gel permeation column(TSKgel G 5000PW+TSKgel G 3000PW, each having a size of 5 mm×30 mm,connected columns). The dissolved components are separated into high andlow molecular fractions by RI (differential refractometer). Eachfraction is subjected to ultrafiltration (fractional molecular weight of5000) for desalting and concentration. The high molecular concentratefraction is passed through a DEAE column (TSKgel DEAE-5PW) followed byelution with 0.2M NaCl. After the eluate is removed, the residue iseluted with 0.5M NaCl followed by ultrafiltration for desalting. Theaqueous solution prepared as above is found to have a potent antitumoractivity. The low molecular concentrate fraction is passed through thesame column to remove water-soluble matters. The eluates obtained byeluting with 0.2M NaCl and 0.5M NaCl are subjected to ultrafiltrationfor desalting. The aqueous solution prepared as above is found to have apotent antitumor activity.

The purified high molecular concentrate fraction is passed through a gelpermeation column as described above. The eluate is desalted byultrafiltration and then concentrated. The substance is found to have amore potent antitumor activity.

(6) The step (5) described above is shown in Table 1 below in moredetail. After the acid degradation, high molecular components (a) andlow molecular components (b) are obtained by gel permeation. Thecomponents (a) and (b) are further subjected to affinity chromatographyto give high molecular component, a-3, low molecular components, b-2 andb-3. The high molecular component, a-3 is further passed through a gelpermeation column to give purified product, H-3. The antitumor-activesubstances of the present invention are the high molecular component,a-3, the low molecular components, b-2 and b-3 and the purified product,H-3.

                                      TABLE 1                                     __________________________________________________________________________     ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                     __________________________________________________________________________

As indicated in Table 1, the profile of chromatogram obtained by gelpermeation (GPC fractionation) is shown in FIG. 1. In FIGS. 2 and 3,profiles obtained when the high and low molecular component fractionsobtained by gel permeation are subjected to affinity chromatography(DEAE fraction) are shown, respectively.

When the high molecular component a-3 shown in Table 1 is subjected togel permeation (GPC fractionation) for further purification, thepurified product H-3 is obtained. The profile of chromatogram by the GPCfractionation is shown in FIG. 4.

(7) With regard to the acid-degraded product, high molecular component(a) and low molecular component (b) obtained by gel permeation, highmolecular components, a-1, a-2 and a-3 and low molecular components,b-1, b-2 and b-3 obtained by affinity chromatography and purifiedproduct H-3, all shown in Table 1, gel permeation analysis wasperformed. The results are shown in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Results of gel permeation analysis                                                         Number-average                                                                        Weight-average                                                                        Dispersion                                                                         Peak                                        Component    molecular weight                                                                      molecular weight                                                                      degree                                                                             maximum                                     __________________________________________________________________________    Acid degraded product                                                                      0.8 × 10.sup.4                                                                  17 × 10.sup.4                                                                   20   27 × 10.sup.4, 1.3 ×                                              10.sup.4                                    High molecular component (a)                                                                10 × 10.sup.4                                                                  35 × 10.sup.4                                                                   3.4  31 × 10.sup.4                         Low molecular component (b)                                                                0.3 × 10.sup.4                                                                  2.7 × 10.sup.4                                                                  8.0  1.2 × 10.sup.4                        High molecular components                                                     a-1           18 × 10.sup.4                                                                  39 × 10.sup.4                                                                   2.4  35 × 10.sup.4                         a-2          8.2 × 10.sup.4                                                                  28 × 10.sup.4                                                                   3.2  26 × 10.sup.4                         a-3          4.0 × 10.sup.4                                                                  29 × 10.sup.4                                                                   7.8  32 × 10.sup.4                         Low molecular components                                                      b-1          0.8 × 10.sup.4                                                                  2.4 × 10.sup.4                                                                  3.0  0.7 × 10.sup.4                        b-2          0.6 × 10.sup.4                                                                  2.4 × 10.sup.4                                                                  4.1  1.7 × 10.sup.4                        b-3          0.6 × 10.sup.4                                                                  2.0 × 10.sup.4                                                                  3.8  1.7 × 10.sup.4                        Purified product H-3                                                                        17 × 10.sup.4                                                                  38 × 10.sup.4                                                                   2.3  30 × 10.sup.4                         __________________________________________________________________________

Conditions for gel permeation analysis:

Column: TSKgel G5000PW +TSKgel G3000PW (each 21.5 mm×300 mm)

Detection: RI

Column temperature: 40° C.

Moving phase: 50 mM sodium nitrate

Flow rate: 0.5 ml/min

Volume loaded: 200 μl (ca. 1 mg/ml, moving phase)

Molecular weight calibration: pullulan

(Shodex: 0.58×10¹, 1.22×10⁴, 2.37×10⁴, 4.80×10⁴, 10.0×10⁴, 18.6×10 ⁴,38.6×10 ⁴, 85.3×10⁴)

The antitumor-active substance of the present invention having anaverage molecular weight of 29×10⁴ daltons and a dispersion degree of7.3 corresponds to the high molecular component, a-3 shown in Tables 1and 2. The antitumor-active substances of the present invention having aweight-average molecular weight of 2.4×10⁴ daltons and a dispersiondegree of 4.1 and having a weight-average molecular weight of 2.0×10⁴daltons and a dispersion degree of 3.6 correspond to the low molecularcomponents, b-2 and b-3, respectively, shown in Tables 1 and 2.

The antitumor-active substance of the present invention having aweight-average molecular weight of 38×10⁴ daltons and a dispersiondegree of 2.3 corresponds to the purified product H-3 obtained bypurifying the high molecular component a-3 shown in Tables 1 and 2through gel permeation, desalting and concentration.

(8) With respect to the purified product H-3 of the present invention,IR was determined by the KBr tablet method. The resulting IR spectrum isshown in Table 5. The spectrum reveals that an absorption appears at 881cm⁻¹. It is assumed that the absorption would be derived fromB-D-gluropyrano bond.

With respect to the purified product H-3, one-dimensional?? NMR spectrum(¹ H-NMR, ¹³ C-NMR) was measured by AC-300P manufactured by BRUKER Co.,Ltd. The measurement temperature was 298K. The one-dimensional protonNMR-spectrum is shown in FIG. 6. In the one-dimensional spectrum, peaksare observed at 5.27 ppm and 4.51 ppm in the proton region. It isconsidered from the data shown in literature (Agric. Biol. Chem., 54,2889 (1990)) that the peak at 5.27 ppm would be derived from1-4-α-glucan and the peak at 4.51 ppm would be from 1-6-β-glucan. Theintegrated data reasonably indicates that a ratio of 1-4-α-glucan to1-6-β-glucan is 4:1.

With respect to the purified product H-3, its optical rotation wasdetermined at 20° C. and a layer length?? of 50 mm in a concentration of1% in 1% sodium hydroxide aqueous solution, using D-line of sodiumspectrum. The specific rotary power was found to be [α]²⁰ _(D) +121°.

(9) With respect to the purified product H-3, all sugars were determinedby the phenol-sulfate method. The sugar content (%) in the purified H-3product was 90.0%.

Furthermore, a sample (5 ml) from the purified H-3 product washydrolyzed (100° C., 3 hours) with 2M trifluoroacetic acid (2 ml). Afterdistillation in vacuo, the constituent sugars were determined using anamino column (CAPCELL PAK, NH₂ (4.6 mm×250 mm)). Only glucose wasdetected.

(10) The protein content of the purified H-3 product was determined bythe Lowry method. Bovine albumin was used as a standard. The proteincontent (%) of H-3 was found to be 3.4%.

After 8 mg of the purified H-3 product was hydrolyzed (110° C., 22hours) with 6N hydrochloric acid (2 ml), its constituent amino acidswere determined using HPLC (Hitachi, Model L-8500 type amino acidanalyzer). The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Composition of Amino Acids                                                             Compositional ratio                                                           (% mol/mol)                                                                   Purified H-3 Product                                                 ______________________________________                                        Asx        10.4                                                               Thr        5.7                                                                Ser        7.6                                                                Glx        11.4                                                               Gly        9.3                                                                Ala        10.3                                                               Val        6.6                                                                (Cys)2     0.3                                                                Met        1.2                                                                Ile        5.0                                                                Leu        9.3                                                                Tyr        1.8                                                                Phe        4.0                                                                Lys        5.4                                                                His        1.6                                                                Arg        4.5                                                                Pro        5.6                                                                Total      100.0                                                              ______________________________________                                    

The glucosamine content (%) detected was 0.06%.

EXAMPLE 2

Assay for antitumor activity

Meth-A (derived from fibrosarcoma) was simultaneously inoculated to mice(5 mice in one group, BALB/c, male mice of 6 weeks old) intradermally atthe right flank (1×10⁶) and left flank (2×10⁵) On Days 3, 4 and 5 afterthe inoculation, each of physiological saline solutions of the highmolecular component a-3 and the low molecular components b-2 and b-3obtained in Example 1, which are the antitumor-active substances of thepresent invention, was injected into the tumor at the same right flank(not injected into the tumor at the left flank) in a dose of 1 mg dryweight/mouse. For control, physiological saline alone was injected intothe tumor at the right flank in another group of mice (control). Forcomparison in effects, the acid-degraded product prior to purificationwas injected into the right tumor in a further group of mice, followingthe schedule above.

A size (area) and weight of the tumor at the right flank and the leftflank were measured at definite intervals up to Day 21 after the tumorinoculation, whereby the effects of the extracts obtained in Example 1were examined.

The experimental results on Day 21 after the tumor inoculation are shownin Tables 4 and 5.

                                      TABLE 4                                     __________________________________________________________________________    Antitumor effect of the high molecular component                                         Tumor free/                                                                         Tumor Size                                                                              %    Tumor Weight                                                                         %                                                 Total (mm.sup.2)                                                                              inhibition                                                                         (g ± S.D.)                                                                        inhibition                             __________________________________________________________________________    High molecular                                                                        Right                                                                            0/5    242.4 ± 97.37*                                                                      52.5 1.4 ± 0.65                                                                        60.0                                   component (a-1)                                                                       Left                                                                             0/5    105.2 ± 93.26*                                                                      63.0 0.5 ± 0.57                                                                        68.8                                   High molecular                                                                        Right                                                                            1/5     96.0 ± 162.72*                                                                     61.6 1.3 ± 1.12                                                                        62.9                                   component (a-2)                                                                       Left                                                                             2/5     114.8 ± 132.82**                                                                   56.9 0.7 ± 0.92                                                                        56.3                                   High molecular                                                                        Right                                                                            3/5    25.4 ± 31.11*                                                                       95.1 0.1 ± 0.15                                                                        97.1                                   component (a-3)                                                                       Left                                                                             3/5    25.2 ± 35.27*                                                                       91.1 0.6 ± 0.44                                                                        62.5                                   Acid degraded                                                                         Right                                                                            0/5    265.0 ± 71.27*                                                                      48.0 1.6 ± 0.57                                                                        53.3                                   product Left                                                                             0/5    149.2 ± 82.64*                                                                      47.6 0.8 ± 0.45                                                                        50.0                                   Control Right                                                                            0/5   510.0 ± 58.74                                                                        N/A  3.5 ± 0.52                                                                        N/A                                            Left                                                                             0/5   284.4 ± 70.39                                                                        N/A  1.6 ± 0.32                                                                        N/A                                    __________________________________________________________________________     Note: When compared to control,                                               *significant difference at p < 0.01                                           **significant difference at p < 0.05                                     

                                      TABLE 5                                     __________________________________________________________________________    Antitumor effect of the low molecular component                                          Tumor free/                                                                         Tumor Size                                                                             %    Tumor Weight                                                                         %                                                  Total (mm.sup.2)                                                                             inhibition                                                                         (g ± S.D.)                                                                        inhibition                              __________________________________________________________________________    Low molecular                                                                         Right                                                                            0/5    470.2 ± 125.34.sup.+                                                                5.3 3.1 ± 1.03                                                                        16.2                                    component (b-1)                                                                       Left                                                                             0/5    271.4 ± 87.90*                                                                     17.1 1.9 ± 1.19                                                                        24.0                                    Low molecular                                                                         Right                                                                            1/5    131.8 ± 75.11*                                                                     73.4 0.7 ± 0.39                                                                        81.0                                    component (b-2)                                                                       Left                                                                             1/5    143.2 ± 111.42*                                                                    56.3 0.9 ± 0.68                                                                        54.0                                    Low molecular                                                                         Right                                                                            1/5    81.4 ± 45.78*                                                                      83.6 0.5 ± 0.27                                                                        86.5                                    component (b-3)                                                                       Left                                                                             1/5    115.6 ± 117.33*                                                                    64.7 0.7 ± 0.82                                                                        72.6                                    Acid degraded                                                                         Right                                                                            0/5    288.8 ± 164.75*                                                                    41.8 1.9 ± 1.24                                                                        48.6                                    product Left                                                                             3/5    74.4 ± 91.20*                                                                      77.3 0.3 ± 0.50                                                                        88.0                                    Control Right                                                                            0/5    496.4 ± 104.22                                                                     N/A  3.7 ± 0.75                                                                        N/A                                             Left                                                                             0/5   327.4 ± 51.91                                                                       N/A  2.5 ± 0.76                                                                        N/A                                     __________________________________________________________________________     Note: When compared to control,                                               *significant difference at p < 0.01                                           **significant difference at p < 0.05                                     

The results shown in Tables 4 and 5 reveal that the high molecularcomponent a-3 and the low molecular components, b-2 and b-3 of thepresent invention exhibit a potent antitumor activity. When compared tocontrol, the tumor growth at the left flank was also inhibited, thoughthese components were not injected there. The results thus reveal thatthe substances of the present invention activated immunocompetent cellsof host and the antitumor activity was exerted by the so activatedimmune function.

EXAMPLE 3

Assay for antitumor activity

Meth-A tumor cells (fibrosarcoma) were simultaneously inoculated on mice(5 mice in one group, BALB/c, male mice of 6 weeks old) intradermally atthe right flank (1×10⁶) and left flank (2×10⁵). On Days 3, 4 and 5 afterthe inoculation, the antitumor-active substance (purified product, H-3)of the present invention obtained in Example 1 was injected into thetumor at the right flank, in the same schedule. For comparison ineffects, the acid-degraded product prior to purification was alsoinjected into the right tumor in another group of mice in the sameschedule. For control, physiological saline alone was injected into thetumor at the right flank in another group of mice. A size (area, mm²) ofthe tumor at the right flank and the left flank was measured every otherday up to Day 21 after the tumor inoculation, whereby the effects of theextracts obtained in Example 1 were examined. On Day 21, animals weresacrificed to weigh the tumors (g). The results of the tumor weight andsize on Day 21 are shown in Table 6. In FIGS. 7 and 8, the experimentalresults obtained at the right and left flanks up to Day 21 after thetumor inoculation are shown, respectively. As is clearly noted fromTable 6 and FIGS. 7 and 8, the antitumor-active substances of thepresent invention exhibit a potent antitumor activity against Meth-Atumor cells.

                                      TABLE 6                                     __________________________________________________________________________               Tumor free/                                                                         Tumor Size                                                                              %    Tumor Weight                                                                          %                                     Antitumor-active                                                                         Total (mm.sup.2 ± S.D.)                                                                    inhibition                                                                         (g ± S.D.)                                                                         inhibition                            __________________________________________________________________________    Purified product                                                                      Right                                                                            5/5    0 ± 0*.sup.+                                                                        100.0                                                                               0 ± 0*.sup.+                                                                      100.0                                 H-3     Left                                                                             4/5     2.4 ± 4.80*.sup.+                                                                  98.2 <0.1*.sup.++                                                                          ≧100.0                         Acid degraded                                                                         Right                                                                            0/5    125.4 ± 77.55**                                                                     55.7   0.8 ± 0.56**                                                                     70.4                                  product Left                                                                             2/5     55.6 ± 56.95***                                                                    58.8   0.3 ± 0.30**                                                                     70.0                                  Control Right                                                                            0/5   282.8 ± 88.38                                                                        N/A   2.7 ± 1.15                                                                        N/A                                           Left                                                                             0/5   135.0 ± 57.12                                                                        N/A   1.0 ± 0.52                                                                        N/A                                   __________________________________________________________________________     *P < 0.01 vs. Control                                                         **P < 0.01 vs. Control                                                        ***P < 0.05 vs. Control                                                       .sup.+ P < 0.01 vs. aciddegraded fraction 3                                   .sup.++ P < 0.05 vs. aciddegraded fraction 3                                  N/A: not applicable                                                      

EXAMPLE 4

Preparation of tablet

A mixture of 100 g of the antitumor-active substance (high molecularcomponent, a-3) of the present invention obtained in Example 1, 100 g ofmannitol and 100 g of glucose is tableted with a conventional tabletingmachine.

EXAMPLE 5

Preparation of tablet

A mixture of 100 g of the antitumor-active substance (purified product,H-3) of the present invention obtained in Example 1, 100 g of mannitoland 100 g of glucose is tableted with a conventional tableting machine.

EXAMPLE 6

Preparation of health food

After 1 liter of aqueous solution containing 1 mg of theantitumor-active substance (high molecular component, a-3) of thepresent invention obtained in Example 1 is added to a suitable amount ofdextrin, the mixture is adsorbed to a base food.

The resulting powders are extruded into an extrusion granulator througha net of 1 mm. The granules are received on a 12 mesh sieve to sievethrough.

The granules are dried at 60° C. overnight in a drier to give granuleswith about 3% moisture content. The granules are used as an additive totea or the like.

EXAMPLE 7

Preparation of health food

After 1 liter of aqueous solution containing 1 mg of theantitumor-active substance (purified product H-3) of the presentinvention obtained in Example 1 is added to a suitable amount ofdextrin, the mixture is adsorbed to a base food.

The resulting powders are extruded into an extrusion granulator througha net of 1 mm. The granules are received on a 12 mesh sieve to sievethrough.

The granules are dried at 60° C. overnight in a drier to give granuleswith about 3% moisture content. The granules are used as an additive totea or the like.

INDUSTRIAL APPLICABILITY

By degrading with an acid, the hot water-insoluble and ethanol-insolublecomponents of the fruit body of Agaricus blazei Murill belonging to thegenus Agaricus, preferably by extracting the insoluble components withan ammonium oxalate aqueous solution, subjecting the extract to aciddegradation, and then purifying the extract, the substances having apotent antitumor activity against solid cancer can be obtained.

What is claimed is:
 1. An antitumor-active substance selected from thegroup consisting of:a substance having a molecular weight of 38×10⁴Daltons and a dispersion degree of 2.3, a substance having a molecularweight of 29×10⁴ Daltons and a dispersion degree of 7.3, a substancehaving a molecular weight of 2.4×10⁴ Daltons and a dispersion degree of4.1 and a substance having a molecular weight of 2.0×10⁴ Daltons and adispersion degree of 3.6,said substance being obtainable by purificationof the acid degraded product of the hot water-insoluble andethanol-insoluble fraction of the fruit body of Agaricus blazei Murillbelonging to the genus Agaricus.
 2. The antitumor-active substanceaccording to claim 1, which is obtainable by extracting the hotwater-insoluble and ethanol-insoluble fraction of said fruit body withan ammonium oxalate aqueous solution, subjecting the extract to aciddegradation and then purifying the acid-degraded product.
 3. Theantitumor-active substance according to claim 2, which has been purifiedby means of gel permeation followed by affinity chromatography.
 4. Theantitumor-active substance according to claim 2, wherein saidantitumor-active substance has a weight-average molecular weight of29×10⁴ Daltons and a dispersion degree of 7.3, and is mainly composed of1-4-p-glucan and 1-6-β-glucan in a ratio of 4:1.
 5. An antitumorcomposition comprising as an active ingredient an effective amount ofthe antitumor-active substance according to claim 2 in combination witha pharmaceutically acceptable carrier.
 6. A health food comprising as anactive ingredient an effective amount of an antitumor-active substanceaccording to claim
 2. 7. The antitumor-active substance according toclaim 1, which has been purified by means of gel permeation followed byaffinity chromatography.
 8. The antitumor-active substance according toclaim 7, wherein said antitumor-active substance has a weight-averagemolecular weight of 29×10⁴ Daltons and a dispersion degree of 7.3, andis mainly composed of 1-4-α-glucan and 1-6-β-glucan in a ratio of 4:1.9. An antitumor composition comprising as an active ingredient aneffective amount of the antitumor-active substance according to claim 7in combination with a pharmaceutically acceptable carrier.
 10. A healthfood comprising as an active ingredient an effective amount of anantitumor-active substance according to claim
 7. 11. A health foodcomprising as an active ingredient an effective amount of anantitumor-active substance according to claim
 7. 12. Theantitumor-active substance according to claim 1 wherein saidantitumor-active substance has a weight-average molecular weight of29×10⁴ Daltons and a dispersion degree of 7.3 and is mainly composed of1-4-α-glucan and 1-6-β-glucan in a ratio of 1:4.
 13. An antitumorcomposition comprising as an active ingredient an effective amount ofthe antitumor-active substance according to claim 12 in combination witha pharmaceutically acceptable carrier.
 14. An antitumor compositioncomprising as an active ingredient, an effective amount of theantitumor-active substance in combination with a pharmaceuticallyacceptable carrier.
 15. A health food comprising as an activeingredient, an effective amount of the antitumor-active substanceaccording to claim 1.